Process for immobilized nano-sized metal particles

ABSTRACT

The present invention provides a new process using biological method for the preparation of immobilized nano-particles of metals. Fungi are used to efficiently prepare immobilized nano-particles of various metals ions such as Au, Ag, Pd, Pt, Ni, Rh and Ru from their aqueous solutions.

FIELD OF THE INVENTION

[0001] The present invention relates to a process for preparingimmobilized nano- sized metal particles. More particularly, the presentinvention relates to a new and improved process employing an efficient,easy and environmentally friendly method for preparing stable,immobilized colloidal nano-particles in aqueous solutions usingnaturally occurring bio-materials such as fungi.

BACKGROUND OF THE INVENTION

[0002] Nano-particles are extremely important materials in differentareas ranging from nano-technology, non-linear optics, diode lasers,smart sensors, markers in drugs, gene sequencing to catalysis.Nano-materials can be obtained by various chemical and physical methods.Some examples of physical methods are vapour deposition, lithographicprocesses and molecular beam epitaxy (MBE). Chemical methods include thepopular borohydride and citrate reduction methods for the preparation ofcolloidal metal (like gold, silver etc.) particles. Reference may bemade to D. A. Handley, Colloidal Gold: Principles, Methods andApplications; Hayat, M. A. Editor, Academic Press, San Diego, Calif.1989; Vol.1, Chapter 2, wherein details of such chemical routes aregiven. Reduction of metal ions by radiolysis is also conventionally usedfor preparing nano-sized metal particles. However, the methods mentionedabove suffer from drawbacks such as being environmentally hazardous(chemical methods) and result in the quick agglomeration ofnano-particles leading to big particles for poor monodispersity.

[0003] Although specific capping agents are used in some of theabovementioned methods to restrict the size of the colloidal metalparticles and to stabilize the particle size distribution, this makesthe whole system quite complicated and user unfriendly. Anotherdisadvantage, particularly of the radiolysis method, is that it is quitecomplicated and gamma ray sources are not readily available.

OBJECTS OF THE INVENTION

[0004] The main object of the invention is to provide an improvedprocess for preparing immobilized nano-sized metal particles using anenvironment friendly biological method.

[0005] Another object of the invention is to provide a process whichuses naturally occurring fungi under aqueous medium.

[0006] Another object of the invention is to provide the process forpreparation of nano-sized metal particles, which are deposited on to thefungus cell wall.

[0007] Another object of the invention is to provide a process where theformation of nano-particles occurs on the surface of biomass and not inthe solution.

SUMMARY OF THE INVENTION

[0008] Accordingly the present invention provides an improved processfor preparing immobilized nano-sized metal particles, which comprisestreating wet fungal mycelia with a metal ion solution at temperature inthe range of 15 to 40° C. for a period in the range of 2 to 120 hours,separating the biomass to obtain the immobilized nano-sized metalparticles deposited on to the surface of the fungal cells.

[0009] In an embodiment of the present invention the wet fungal myceliais obtained by growing the Verticillium (AAT-TS-4) in a culture mediumfor a period of 2 to 120 hours at temperature ranging between 15-40° C.under aseptic conditions, separating the biomass by centrifugation,washing several times with sterile water, and then incubating the wholereaction mixture at 15 to 40° C. and atmospheric pressure.

[0010] In another embodiment the metal ion solution is obtained bydissolving metal salts of group IB-VIIIB metals in water.

[0011] In a further embodiment of the invention, the metal is selectedfrom the group consisting of Au, Ag, Pd, Pt, Ni Rh and Ru.

[0012] In a further embodiment of the invention, the metal salts areselected from the group consisting of halides, nitrates and carbonates.

[0013] In another embodiment the metal ion solution is obtained bydissolving the acidic form of metals in water.

[0014] In a further embodiment of the invention, the acidic form of themetal is selected from chloroauric acid and chloroplatinic acid.

[0015] In another embodiment of the invention concentration of metalions per gram of wet fungal mycelia is in the range of 10 to 200 mgmetal ions per gram of wet fungal mycelia.

[0016] In another embodiment of the invention concentration of metalions per gram of wet fungal mycelia is in the range of 10 to 100 mgmetal ions per gram of wet fungal mycelia.

[0017] In another embodiment of the invention concentration of metalions per gram of wet fungal mycelia is in the range of 25 to 100 mgmetal ions per gram of wet fungal mycelia.

[0018] In yet another embodiment of the invention ratio of water to wetfungal mycelia is 100:1 (w/w)

[0019] In another embodiment of the invention the fungus Verticilliumdesignated as AAT-TS-4 is taken as whole cell as wet solid mass.

[0020] In another embodiment of the invention, reaction of the fungusand a source of metal ions in solution is carried out in water.

[0021] In another embodiment of the invention the incubation/reactiontemperature is in the range of 15-40° C., preferably 23-33° C., mostpreferably 25-29° C.

[0022] The process for the present invention is described herein belowwith examples that are illustrative and should not be construed to limitthe scope of the present invention.

EXAMPLE 1

[0023] In this experiment, 10 g of wet fungal mycelia (VerticilliumAAT-TS-4), grown in a culture medium, separated from medium bycentrifugation, washed several times with water through centrifugation,was taken in a autoclaved conical flask and then 100 ml solution of 100mg of HAuCl₄ in water was added.

[0024] The conical flask was then plugged with cotton and incubated at27° C. Samples were collected periodically by filtration of solutioncontaining the fungus inside the inoculation chamber under laminar flowcondition. Bio-transformation was routinely monitored by visualinspection of the biomass as well as measurement of UV-vis spectra fromthe fungal cells.

[0025] Films of the fungal cells (both before and after exposure to Au⁺ions for 72 h) for UV-vis spectroscopy and scanning electron microscopy(SEM) studies were prepared by solution casting fungal cells onto Si(111) wafers and thoroughly drying film in flowing N₂. UV-visspectroscopy measurement of films were made on a Shimadzu dual-beamspectrophotometer (model UV-1601PC) operating the reflection mode at aresolution of 2 nm.

[0026] These data confirm the presence of gold nano-particles on to thesurface of the biomaterial. UV-vis spectra of the clear aqueous solutionafter reaction with the mycelial cells for 72 h showed the absence ofthe characteristic plasmon resonance band of gold ca 433 nm indicatingthe absence of gold in the solution.

EXAMPLE 2

[0027] In this experiment, 10 g of wet fungal mycelia (Verticillium),grown in a culture medium, separated from medium by centrifugation,washed several times with water through centrifugation, was taken in anautoclaved conical flask and then a solution containing 25 mg of HAuCl₄in 100 ml water was added.

[0028] The conical flask was then plugged with cotton and incubated at27° C. Samples were collected periodically by filtration of solutioncontaining the fungus inside the inoculation chamber under laminar flowcondition. Bio-transformation was routinely monitored by visualinspection of the biomass as well as measurement of the UV-vis spectrafrom the fungal cells.

[0029] Films of the fungal cells (both before and after exposure to Au⁺ions for 72 h) for UV-vis spectroscopy and scanning electron microscopy(SEM) studies were prepared by solution casting the fungal cells onto Si(111) wafers and thoroughly drying the film in flowing N₂. UV-visspectroscopy measurements of the films were made on a Shimadzu dual-beamspectrophotometer (model UV-1601PC) operating in reflection mode at aresolution of 2 nm.

[0030] These data confirm the presence of gold nanoparticles on thesurface of the biomaterial. UV-vis spectra of the clear aqueous solutionafter reaction with the mycelial cells for 72 h showed the absence ofthe characteristic plasmon resonance band of gold ca 533 nm indicatingthe absence of gold in the solution.

EXAMPLE 3

[0031] In this experiment, 10 g of wet fungal mycelia (Verticillium),grown in a culture medium, separated from medium by centrifugation,washed several times with water through centrifugation, was taken in anautoclaved conical flask and then 250 mg of HAuCl₄ in 100 ml water wasadded.

[0032] The conical flask was then plugged with cotton and incubated at27° C. Samples were collected periodically by filtration of solutioncontaining the fungus inside the inoculation chamber under laminar flowcondition. Bio-transformation was routinely monitored by visualinspection of the biomass as well as measurement of the UV-vis spectraform the fungal cells.

[0033] Films of fungal cells (both before and after exposure to Au⁺ ionsfor 72 h) for UV-vis spectroscopy and scanning electron microscopy (SEM)studies were prepared by solution casting fungal cells onto Si(111)wafers and thoroughly drying the film in flowing N₂. UV-vis spectroscopymeasurements of the films were made on a Shimadzu dual-beamspectrophotometer (model UV-1601IPC) operating in reflection mode at aresolution of 2 nm.

[0034] These data confirm the presence of gold nano-practicles on thesurface of the biomaterial. UV-vis spectra of the clear aqueoussolutions after reaction with the mycelial cells for 72 h showed theabsence of the characteristic plasmon resonance band of gold ca 533 nmindicating the absence of gold in the solution.

EXAMPLE 4

[0035] In this experiment, 10 g of wet fungus (Verticillium) grown in aculture medium, separated from medium by centrifugation, washed severaltimes with water through centrifugation, was taken in an autoclavedconical flask and then 125 mg AgNO₃ in 100 water was added.

[0036] The conical flask was then plugged with cotton and incubated at27° C. Samples were collected periodically by filtration of solutioncontaining the fungus inside the inoculation chamber under laminar flowcondition. Presence of nano sized Ag particles deposited on to thefungal cells was confirmed by evolution of plasmon resonance band around400 nm. The brown coloration is a clear indication of formation ofsilver nano-clusters. The range of the silver nano-particles size wasfound to be 5-80 nm.

EXAMPLE 5

[0037] In this experiment, 10 g of wet fungal mycelia (Verticillium),grown in a culture medium, separated from medium by centrifugation,washed several times with water through centrifugation, was taken in aautoclaved conical flask and then 50 mg AgNO₃ in 100 ml water was added.

[0038] The conical flask was then plugged with cotton and incubated at37° C. Samples were collected periodically by filtration of solutioncontaining the fungus inside the inoculation chamber under laminar flowcondition. Samples were collected between 1 and 86^(th) and each samplewas characterized by UV-vis spectroscopy fluorescence spectroscopy, TEManalysis. Evolution of plasmon resonance band around 400 nm and thebrown coloration is a clear indication of formation of silvernanoclusters. The range of the silver nanoparticles size was found to beca. 50 nm.

EXAMPLE 6

[0039] In this experiment, 10 g of wet fungal mycelia (Verticillium)grown, in a culture medium, separated from medium by centrifugation,washed several times with water through centrifugation, was taken in anautoclaved conical flask and then 100 ml solution containing 100 mgNi(NO₃)₂ (nickel nitrate) was added. The conical flask was then pluggedwith cotton and incubated at 22° C. Samples were collected periodicallyby filtration of solution containing the fungus inside the inoculationchamber under lamiar flow condition Samples were collected at 86 h andcharacterized by UV-vis spectroscopy, TEM analysis and by fluorescencespectroscopy. Evolution of plasmon resonance band around 415 nm is clearindication of formation of Ni-nano-clusters in solution. Sizes of thenano-clusters were determined by TEM analysis and found to be 100 nm.

EXAMPLE 7

[0040] In this experiment, 10 g of wet fungal mycelia (Verticillium)grown in a culture medium, separated from medium by centrifugation,washed several times with water through centrifugation, was taken in anautoclaved conical flask and then 25 mg NiSO₄ in 100 ml water was added.

[0041] The conical flask was then plugged with cotton and incubated at25° C. Samples were collected periodically by filtration of solutioncontaining the fungus inside the inoculation chamber under laminar flowcondition. The samples were collected between 1 and 96 h and each stagewas characterized by UV-vis spectroscopy fluorescence spectroscopy, TEManalysis. The brown coloration of the fungal mycelial biomass, evolutionof the plasmon resonance band around 415 nm and TEM analysis indicatedthe formation of nickel nanoclusters, deposited on to the fungal cell,in the range of 50-100 nm. No evidence of the presence of Ni in thesolution was observed.

EXAMPLE 8

[0042] In this experiment, 10 g of wet fungal mycelia (Verticillium),grown in a culture medium, separated from medium by centrifugation,washed severed times with water through centrifugation, was taken in anautoclaved conical flask and then 100 ml aqueous solution containing 125mg of H₂PtCl₆ (chloroplatinic acid) in water were added.

[0043] The conical flask was then plugged with cotton and incubated at33° C. Samples were collected periodically by filtration of solutioncontaining the fungus inside the inoculation chamber under laminar flowcondition. Samples were collected between 1 and 96 h and the sample werecharacterized by UV-vis spectroscopy, fluorescence spectroscopy, TEManalysis. The evolution of the plasmon resonance band at 215 nm is aclear indication of the formation of Pt-nano-particles deposited on tofungal cell. The samples were further characterized by TEM analysis andthe particle size was found to be in the range of 30-50 nm. No evidencecould be obtained for the presence of Pt. in solution after thereaction.

[0044] It is therefore clear that the present invention provides a newprocess using biological method for the preparation of immobilized nanoparticles of metals obviating the drawbacks of the prior art methods.The process of the present invention describes a new biological method,instead of chemical or physical methods for preparing immobilized metalparticles. This is the first time that fungi are used to efficientlyprepare immobilized nano-particles of various metals ions from theiraqueous solutions.

JUSTIFICATION AND ADVANTAGES OF THE PRESENT INVENTION

[0045] The use of naturally occurring fungi under aqueous medium.

[0046] The immobilized nano-sized metal particles are stable.

[0047] The method of the invention is simple and environmentallyfriendly.

[0048] The formation of nano-particles occurs on the surface, thereforeimmobilizing them and the metal nano-particles are not released in tothe solution.

[0049] A single step method for obtaining immobilized nano-particles ofmetals.

1. A process for preparing immobilized nano-sized metal particlescomprising treating wet fungal mycelia of Verticillium (AAT-TS-4) with ametal ion solution at temperature in the range of 15 to 40° C. for aperiod in the range of 2 to 120 hours, separating the biomass to obtainthe immobilized nano-sized metal particles deposited on to the surfaceof the fungal cells.
 2. A process as claimed in claim 1 wherein the wetfungal mycelia is obtained by growing the Verticillium (AAT-TS-4) in aculture medium for a period of 2 to 120 hours at temperature rangingbetween 15-40° C. under aseptic conditions, separating the biomass bycentrifugation, washing several times with sterile water, and thenincubating the whole reaction mixture at 15 to 40° C. and atmosphericpressure.
 3. A process as claimed in claim 1 wherein the metal ionsolution is obtained by dissolving metal salts of group IB-VIIIB metalsin water.
 4. A process as claimed in claim 3 wherein the metal isselected from the group consisting of Au, Ag, Pd, Pt, Ni, Rh and Ru. 5.A process as claimed in claim 3 wherein the metal salts are selectedfrom the group consisting of halides, nitrates and carbonates.
 6. Aprocess as claimed in claim 1 wherein the metal ion solution is obtainedby dissolving the acidic form of metals in water.
 7. A process asclaimed in claim 6 wherein the acidic form of the metal is selected fromchloroauric acid and chloroplatinic acid.
 8. A process as claimed inclaim 1 wherein the concentration of metal ions per gram of wet fungalmycelia is in the range of 10 to 200 mg metal ions per gram of wetfungal mycelia.
 9. A process as claimed in claim 8 wherein theconcentration of metal ions per gram of wet fungal mycelia is in therange of 10 to 100 mg metal ions per gram of wet fungal mycelia.
 10. Aprocess as claimed in claim 8 wherein the concentration of metal ionsper gram of wet fungal mycelia is in the range of 25 to 100 mg metalions per gram of wet fungal mycelia.
 11. A process as claimed in claim 1wherein the ratio of water to wet fungal mycelia is 100:1 (w/w).
 12. Aprocess as claimed in claim 1 wherein the fungus Verticillium AAT-TS-4is taken as whole cell as wet-solid mass.
 13. A process as claimed inclaim 1 wherein the reaction of the fungus and metal ion source insolution is carried out in water.
 14. A process as claimed in claim 1wherein the incubation/reaction temperature is in the range of 15-40° C.15. A process as claimed in claim 14 wherein the incubation/reactiontemperature is in the range of 23-33° C.
 16. A process as claimed inclaim 14 wherein the incubation/reaction temperature is in the range of25-29° C.